Method for producing novel spinosyn derivatives

ABSTRACT

The present invention relates to methods for producing novel spinosyn derivatives which are substituted with a 1-hydroxy-ethyl radical in the C-21 position and to novel spinosyn derivatives of this type per se and to their use for producing novel spinosyns.

[0001] The present invention relates to methods for producing novelspinosyn derivatives which are substituted with a 1-hydroxy-ethylradical in the C-21 position and to novel spinosyn derivatives of thistype per se and to their use for producing novel spinosyns.

[0002] The spinosyns are known compounds. Spinosyns are fermentationproducts which are produced by cultures of the actinomycetesSaccharopolyspora spinosa. Natural spinosyns consist of a tetracyclicpolyketide backbone (aglycone) with a 12-membered macrolide ring and a5,6,5-cis-anti-trans-tricycle and also a D-forosamine and a2,3,4-tri-O-methyl-L-rhamnose sugar moiety (Kirst et al., 1991,Tetrahedron Letters, 32:4839). More than 20 different natural spinosyns,the “A83543 complex”, have been described previously (cf. WO 97/00265,WO 94/20518 and WO 93/09126). EP-A 375316, for example, describes thespinosyns A, B, C, D, E, F, G, H and J. WO 93/09126 discloses thespinosyns L, M, N, Q, R, S and T. The spinosyns K, 0, P, U, V, W and Yand their derivatives are mentioned in WO 94/20518. These compounds varyin the substitution of one or more methyl groups on the tetracyclicbackbone, on the D-forosamine sugar moiety or on the2,3,4-tri-O-methyl-L-rhamnose sugar moiety. A 17-pseudoaglycone whichreacts the D-forosamine sugar moiety has likewise been isolated from S.spinosa culture broth.

[0003] The main components of the A83543 complex produced by S. spinosaare the variants spinosyn A and spinosyn D which are the essentialcomponents of the product spinosad (cf. Pesticide Manual, British CropProtection Council, 11^(th) Ed., 1997, page 1272 and Dow Elanco TradeMagazine Down to Earth, Vol. 52, NO. 1, 1997 and the literature quotedtherein).

[0004] If the amino sugar in the C-17 position is not present, thecompounds are referred to as spinosyn A, D, etc. 17-pseudoaglycone; ifthe neutral sugar in the C-9 position is not present, the compounds arereferred to as spinosyn A, D, etc. 9-pseudoaglycone. Spinosyns withoutthe two sugar residues in the positions C-9 and C-17 are referred to asspinosyn aglycone.

[0005] Spinosyns are suitable for controlling arachnids, nematodes,ectoparasites (cf. WO 01/11962, WO 01/11963, WO 01/11964) and insects,in particular Lepidoptera and Diptera species. In addition, technicalapplication of the spinosyns is environmentally sound and, moreover,this substance class has an attractive toxicological profile.

[0006] However, animal pests, in particular ectoparasites, or plantpests which are currently controlled using spinosyns can be expected tobe able to develop a resistance to these commercially available activesubstances. It is therefore important to produce novel biologicallyactive spinosyn derivatives which can replaced the spinosyns currentlyused for controlling pests.

[0007] Recently, Saccharopolyspora sp. (LW107129) has been used togenerate specific natural aglycone derivatives of spinosyn which have ahydroxyl group in the C-8 position of the macrolide backbone and whichhave become known as insecticidal compounds (cf. WO 0.01/19840). Whilenumerous modifications of spinosyns have been carried out (cf. WO97/00265), derivatizations on the methyl group and ethyl group in theC-21 position of the macrolide backbone drew only little attention.Although functionalization of the alkyl radical in the C-21 positionwould be advantageous, inter alia for derivatization reactions, only fewspinosyn derivatives are known whose substituents have a hydroxyl groupin C-21. For example, only recently have spinosyn derivatives beendescribed which are substituted in C-21 with a 3-hydroxy-1-butenylradical, which may, where appropriate, additionally carry a hydroxylgroup in the abovementioned C-8 position and which have an insecticidalaction (cf. WO 01/19840).

[0008] While chemical synthesis methods for preparing spinosynderivatives have been described (cf. Martynow, J. G. and Kirst, H. A.,1994, J. Org. Chem., 59: 1548), there has up until now been no knowledgeabout chemical synthesis of the above-mentioned spinosyn derivativeshaving a 1-hydroxy-ethyl radical in the C-21 position.

[0009] It is the object of the present invention to provide suitablemethods which may be used for the selective and/or stereospecificproduction of novel spinosyn derivatives having a 1-hydroxy-ethylradical in the C-21 position.

[0010] The object was achieved by providing methods for producingcompounds of the general formula (I),

[0011] in which

[0012] A-B is any of the following groups: —HC═CH—, —HC═C(CH₃)—,—H₂C—CH₂— or —H₂C—CH(CH₃)—;

[0013] D is the group

[0014] R¹ is hydrogen or an amino sugar and

[0015] R² is hydrogen or a sugar,

[0016] in which compounds of the general formula (II),

[0017] in which

[0018] A-B, D and R¹ are as defined above,

[0019] are contacted with a microorganism in an aqueous nutrient mediumunder aerobic conditions or with an enzyme extract prepared therefrom orwith one or more enzymes isolated therefrom.

[0020] The starting compounds are thus selectively and/orstereospecifically converted to spinosyn derivatives substituted in theC-21 position by a 1-hydroxy-ethyl radical by means of biotransformationusing microorganisms or their enzymes.

[0021] The term “spinosyn derivatives”, as used herein, also comprisesspinosyn aglycone compounds, i.e. compounds which have the macrolidebackbone of the spinosyns but no sugar radicals.

[0022] Preference is given to using as starting compounds thosecompounds of the general formula (II)

[0023] in which, in the case (1) that

[0024] A-B is any of the following groups: —HC═CH—, —HC═C(CH₃)—,—H₂C—CH₂— or —H₂C—CH(CH₃)— and

[0025] D is the group

[0026] R¹ is an amino sugar of the formula 1a

[0027]  and

[0028] R² is a sugar of the formula 2a

[0029] or in which, in the case (2) that

[0030] A-B is the group —HC═CH— or —H₂C—CH₂—and

[0031] D is as defined above,

[0032] R¹ is an amino sugar of the abovementioned formula 1a and

[0033] R² is hydrogen or a sugar of the formula 2b, 2c, 2d, 2e or 2f

[0034] or in which, in the case (3) that

[0035] A-B is any of the following groups: —HC═CH—, —HC—C(CH₃)— or—H₂C—CH₂— and

[0036] D is as defined above,

[0037] R¹ is hydrogen or an amino sugar of the formula 1b

[0038]  and

[0039] R² is hydrogen or a sugar of the abovementioned formula 2a

[0040] or in which, in the case (4) that

[0041] A-B is the group —HC═CH— or —HC═C(CH₃)— and

[0042] D is as defined above,

[0043] R¹ is an amino sugar of the abovementioned formula 1a and

[0044] R² is a sugar of the formula 2g, 2h, 2i, 2j or 2k

[0045] or in which, in the case (5) that

[0046] A-B is the group —HC═CH— or —H₂C—CH₂— and

[0047] D is as defined above,

[0048] R¹ is an amino sugar of the abovementioned formula 1a and

[0049] R² is a sugar of the formula 2l or 2m

[0050] or in which, in the case (6) that

[0051] A-B is the group —HC═CH— or —HC═C(CH₃—)— and

[0052] D is as defined above,

[0053] R¹ is hydrogen or an amino sugar of the abovementioned formula 1bor an amino sugar of the formula 1c

[0054]  and

[0055] R² is a sugar of the abovementioned formula 2b, 2c, 2g or 2h or asugar of the formula 2n

[0056] or in which, in the case (7) that

[0057] A-B is the group —HC═CH— and

[0058] D is as defined above,

[0059] R¹ is an amino sugar of the abovementioned formula 1a and

[0060] R² is a sugar of the formula 2o

[0061] or in which, in the case (8) that

[0062] A-B is the group —HC═CH— and

[0063] D is as defined above,

[0064] R¹ is an amino sugar of the abovementioned formula 1b and

[0065] R² is a sugar of the abovementioned formula 2d, 2i or 2j

[0066]  or a sugar of the formula 2p

[0067] or in which, in the case (9) that

[0068] A-B is the group —HC═CH— and

[0069] D is as defined above,

[0070] R¹ is hydrogen or an amino sugar of the abovementioned formula 1cand

[0071] R² is a sugar of the abovementioned formula 2i or 2p,

[0072] or in which, in the case (10) that

[0073] A-B is the group —HC═CH— and

[0074] D is as defined above,

[0075] R¹ is an amino sugar of the formula 1d, 1e or 1f

[0076]  and

[0077] R² is a sugar of the abovementioned formula 2a

[0078] or in which, in the case (11) that

[0079] A-B is the group —HC═CH— and

[0080] D is the group

[0081] R¹ is hydrogen or an amino sugar of the abovementioned formula1a.

[0082] Particular preference is given to using as starting compoundsthose compounds of the general formula (II)

[0083] in which, in the case (12) that

[0084] A-B is the group —HC═CH— or —HC═C(CH₃)— and

[0085] D is the group

[0086] R¹ is an amino sugar of the formula 1a and

[0087] R² is a sugar of the-formula 2a, 2g or 2h

[0088] or in which; in the case (13) that

[0089] A-B is the group —HC═CH— and

[0090] D is as-defined above,

[0091] R¹ is an amino sugar of the formula 1a and

[0092] R² is hydrogen or a sugar of the formula 2d, 2e, 2l, 2m or 2o

[0093] or in which, in the case (14) that

[0094] A-B is the group —HC═CH— or —HC═C(CH₃)— and

[0095] D is as defined above,

[0096] R¹ is hydrogen or an amino sugar of the formula 1b and

[0097] R² is hydrogen or a sugar of the formula 2a

[0098] or in which A-B, D and R¹ are as defined in the case (11).

[0099] Very particular preference is given to using as startingcompounds compounds of the general formula (II)

[0100] in which, in the case (15) that

[0101] A-B is the group —HC═CH— or —HC═C(CH₃)— and

[0102] D is the group

[0103] R¹ is an amino sugar of the formula 1a and

[0104] R² is a sugar of the formula 2a

[0105] or in which, in the case (16) that

[0106] A-B is the group —HC═CH— and

[0107] D is as defined above,

[0108] R¹ is an amino sugar of the formula 1a and

[0109] R² is hydrogen or a sugar of the formula 2d, 2l or 2m

[0110] or in which A-B, D and R¹ are as defined in the case (11).

[0111] Most preference is given to using as starting compounds compoundsof the general formula (II)

[0112] in which, in the case (17) that

[0113] A-B is the group —HC═CH— or —HC═C(CH₃)— and

[0114] D is the group

[0115] R¹ is an amino sugar of the formula 1a and

[0116] R² is a sugar of the formula 2a

[0117] or in which, in the case (18) that

[0118] A-B is the group —HC—CH— and

[0119] D is as defined above,

[0120] R¹ is hydrogen and

[0121] R² is hydrogen.

[0122] The present invention also relates to the compounds of thegeneral formula (I) in which A-B, D and R¹ are as defined above.

[0123] The abbreviation “Me” used herein represents methyl; theabbreviation “Et” represents ethyl.

[0124] The method of the invention may be used to form the opticallyactive, stereoisomeric forms but also diastereomeric forms of thecompounds of the general formula (I).

[0125] The compounds of the invention of the formula (I) can exist instereoisomeric forms which either behave as image and mirror image(enantiomers) or which do not behave as image and mirror image(diastereomers). The present invention relates to both the enantiomersand the diastereomers and to the respective mixtures thereof. Theracemic forms, as well as the diastereomers can be resolved into thestereoisomerically uniform components in the known manner. Whereappropriate, methods known per se can be used to interconvert saidisomers.

[0126] The compounds of the invention, in which R¹ is an amino sugar ofthe formulae 1a-1e, may form salts. Salts are formed according to thestandard methods for preparing salts. For example, the compounds of theinvention are neutralized with appropriate acids in order to produceacid addition salts. Representatively usable acid addition salts aresalts which form, for example, due to a reaction with other inorganicacids such as, for example, sulfuric acid, hydrochloric acid,hydrobromic acid, phosphoric acid, or organic carboxylic acids such asacetic acid, trifluoroacetic acid, citric acid, succinic acid, lacticacid, formic acid, maleic acid, camphoric acid, phthalic acid, glycolicacid, glutaric acid, stearic acid, salicylic acid, sorbic acid, cinnamicacid, picric acid, benzoic acid, or organic sulfonic acids such asmethanesulfonic acid and para-toluenesulfonic acid, or with basic aminoacids such as aspartic acid, glutamic acid, arginine, or the like.

[0127] The compounds of the general formula (II), which may be used asstarting compounds for the method of the invention, have been described(cf. Creemer L. C. et al., 1998, J. Antibiotics 51 (8): 795-800; SparksT. C. et al., 1998, J. Econ. Entomol. 91 (6): 1277-1283; Sparks T. C. etal., 2000, Pestic. Biochem. Physiol. 6.7 (3): 187-197; Paquette L. A. etal., 1998, J. Am. Chem. Soc. 120 (11): 2553-2563; Evans D. A. et al.,1993, J. Am. Chem. Soc. 115 (11): 4497-4513; Crouse G. D. et al., 2001,Pest. Manag. Sci. 57 (2): 177-185; Creemer L. C. et al., 2000, J.Antibiotics 53 (2): 171-178; Sparks T. C. et al., 2000, Proc.-BeltwideCotton Conf. Vol. 2: 1225-1229) or may be prepared according to themethod described in WO 01/16303. Similarly, the starting compoundsusable for the method of the invention may be obtained starting from theappropriate natural spinosyns.

[0128] When using, for example, the spinosyn A aglycone of the formula(IIa), the method of the invention can be represented by the followingreaction scheme 1:

[0129] Selective and/or stereospecific hydroxylations of naturalproducts and of synthetic compounds by bioconversion usingmicroorganisms or their enzymes have been described in the literature.Use was made, in particular, of cells and/or enzymes of Gram-positivebacteria such as streptomycetes or Gram-negative bacteria such asPseudomonas or fungi such as Fusarium. Examples of microorganismscontaining appropriate enzyme classes such as, for example, P-450monooxygenases are listed in the following table: Organism/enzymeChemical compound class Reference Streptomyces halstedii Hydroxylationof U.S. Pat. No. 5972994 galbonolides A and B Streptomyces sp. MA 7065Hydroxylation of Taxol and U.S. Pat. No. 5756536 cephalomanninesStreptomyces roseochromogenes Hydroxylation of 11-oxo- or U.S. Pat. No.2864837 (Waksman collection 3689), 11β-hydroxy-6α- Streptomyces sp.(ATCC 11009), methylprogesterone to the and Streptomyces 16α-hydroxyderivative which roseochromogenes (ATCC 3347) is then acetylatedNocardioides luteus 6-Hydroxy-7-deoxytaxanes U.S. Pat. No. 6162622Fusarium moniliforme Preparation of 7α-hydroxyl FR-A 2771105 derivativesfrom dehydroepiandrosterone and pregnenolone Beauveria bassiana2-phenoxypropionic acid WO 95/29249 Cunninghamella blakesleenaHydroxylation of avermectin U.S. Pat. No. 4666937 (ATCC 8688a) EP-A194125 Pseudomonas testosteroni ATCC m-Hydroxybenzoate is U.S. Pat. No.4217416 31492 transformed to give 2,3- dihydroxybenzoate Mortierellamaculata Preparation of pravastatin, WO 00/46175 starting from compactinNADPH cytochrome-P450 Hydroxylation of various WO 93/21326 reductase ofplants substrates Bacterium NRRL-B-18737 Preparation of bisphenol alkylU.S. Pat. No. 5132228 alcohols, starting from bisphenol alkanesPseudomonas mendocina kr-1 Bioconversion of a phenyl WO 89/09828monooxygenase genes compound to a phenolic compound Microorganisms orenzymes Preparation of optically active WO 00/296063-hydroxy-pyrrolidines Recombinant Yarrowia lipolytica Bioconversion ofprogesterone WO 00/03008 expressing heterologous to17α-hydroxyprogesterone cytochrome-P450 systemsGeranylgeraniol-18-hydroxylase Bioconversion of U.S. Pat. No. 5879916purified from Croton sublyratus geranylgeraniol to plaunotol

[0130] According to the invention and to the abovementioned reactionscheme 1, it is possible to contact compounds of the general formula (H)in which A-B, D and R¹ are as defined above with a suitablemicroorganism in an aqueous nutrient medium under aerobic conditions andthen to isolate the desired compounds of the general formula (I).

[0131] It is also possible to use, instead of said microorganisms,enzyme extracts and purified enzymes, if appropriate after addition orwith regeneration of the required cofactors, which are obtainable bycommon methods starting from said microorganisms.

[0132] It is in particular also possible to clone genes determining thebiosynthesis of such enzymes and express them in foreign hosts such as,for example, Escherichia coli. Recombinant bacteria of this type may beused immediately for biotransformation. In addition, it is also possibleto use an enzyme extract of such a recombinant cell or a purifiedprotein for biotransformation, where appropriate after addition or withregeneration of the required cofactors.

[0133] Preference is given to using for the method of the invention amicroorganism from the group of actinomycetes, in particular of thegenus Streptomyces.

[0134] Particular preference is given to using for the method of theinvention a strain of the genus Streptomyces djakartensis, Streptomycesgriseofuscus, Streptomyces caelestis, Streptomyces antibioticus,Streptomyces griseus or Streptomyces aureofaciens.

[0135] Very particular preference is given to using for the method ofthe invention a strain having the characteristic features of thefollowing strains: Name Deposition No. Streptomyces djakartensis NRRLB-12103 Streptomyces griseofuscus DSM 40191 Streptomyces caelestis DSM40084 Streptomyces antibioticus ATCC 11891 Streptomyces griseus DSM40937 Streptomyces aureofaciens DSM 46447

[0136] The strains mentioned in the table have been deposited again withthe Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ),Mascheroder Weg 1b, D-38124 Brunswick, Germany, in accordance withrequirements of the Budapest treaty: Name Deposition No. Streptomycesdjakartensis DSM 14327 Streptomyces griseofuscus DSM 14330 Streptomycescaelestis DSM 14328 Streptomyces antibioticus DSM 14329 Streptomycesgriseus DSM 14331 Streptomyces aureofaciens DSM 14332

[0137] The strains are described in more detail in Example 16. It ispossible to use not only the deposited strains per se but also mutantsthereof, as long as these mutants have the characteristic features ofthe deposited strains, i.e. the mutants must still be capable ofcarrying out the bioconversion of the invention.

[0138] The aqueous nutrient medium preferably contains an assimilablecarbon source and an assimilable nitrogen source.

[0139] The compounds of the formula (I) are produced, for example, whenfermenting a strain of the species Streptomyces djakartensis, S.gtiseofuscus, S. caelestis, S. antibioticus, S. griseus or S.aureofaciens in an aqueous nutrient medium under aerobic conditions inthe presence of compounds of the formula (II). The microorganisms aretypically fermented in a nutrient medium containing a carbon source and,where appropriate, proteinaceous material. Preferred carbon sourcesinclude glucose, brown sugar, sucrose, glycerol, starch, corn starch,lactose, dextrin, molasses, etc. Preferred nitrogen sources includecotton seed meal, yeast, autolyzed bakers' yeast, solid milkconstituents, soybean meal, cornmeal, pancreatic or papainic caseinhydrolyzates, solid distillation components, broths of animal peptone,meat and bone fragments, etc. Preference is given to using combinationsof these carbon and nitrogen sources. Trace elements such as, forexample, zinc, magnesium, manganese, cobalt, iron, etc. need not beadded to the culturing medium as long as tapwater and nonpurifiedconstituents are being used as components of the medium.

[0140] Production of the compounds of the general formula (I) may beinduced at any temperature which ensures sufficient growth of themicroorganisms. The temperature is preferably between 21° C. and 32° C.,particularly preferably approximately 28° C. Optimal production of thecompounds of the formula (1) is usually achieved within 2 to 4 daysafter addition of the compounds of the formula (II) to the culture. Thecompounds of the invention may be produced both in shaker bottles and instirred fermenters.

[0141] Preferred growth conditions and media for growing in shakerflasks are described in Examples 2 to 9.

[0142] The compounds of the invention as biotransformation product maybe isolated from the culturing medium by common methods.

[0143] Various methods may be applied in order to isolate and purify thecompounds of the invention from the fermentation broth, such as, forexample, preparative gel chromatography, preparative chromatography onreversed phase or preparative absorption chromatography. The detectionmay be carried out, for example, by UV absorption or mass spectrometry.

[0144] The compounds of the invention may be used for preparingbiologically active, in particular insecticidal and acaricidal,spinosyns. The biological efficacy of particular natural aglyconederivatives of spinosyn, which have a hydroxyl group in the C-8 positionof the macrolide backbone, has recently been described (cf. WO01/19840). Further examples which may be mentioned are spinosynderivatives with a 3-hydroxy-1-butenyl radical in the C-21 position,which have likewise been described recently. Where appropriate, thesespinosyn derivatives may also additionally carry a hydroxyl group in anyof the abovementioned C-8 position and likewise have an insecticidalactivity (cf. WO 01/19840).

[0145] It is advantageous to use suitable protective groups (PG) whenpreparing further spinosyn derivatives from the inventive compounds ofthe general formula (I) in which R¹ is hydrogen and D is the group C═Oor C—O—R² where R² is hydrogen. Known examples of protective groups (PG)for hydroxyl groups are substituted methyl ethers and ethers,substituted ethyl ethers, substituted benzyl ethers, silyl ethers,esters, carbonates or sulfonates (cf. Greene T. W., Wuts P. G. W. inProtective Groups in Organic Synthesis; John Wiley & Sohns, Inc. 1999,Protection for the hydroxyl group, including 1,2- and 1,3-diols).

[0146] Examples of protective groups (PG) of the substituted methylether type, which may be mentioned, are: methoxymethyl (MOM) ethers,methylthiomethyl (MTM) ethers, (phenyl-dimethylsilyl)methoxymethyl(SMOM-OR) ethers, benzyloxymethyl (BOM-OR) ethers,para-methoxybenzyloxymethyl (PMBM-OR) ethers, para-nitrobenzyloxy-methylethers, ortho-nitrobenzyloxymethyl (NBOM-OR) ethers,(4-methoxyphenoxy)-methyl (p-AOM-OR) ethers, guaiacolmethyl (GUM-OR)ethers, tert-butoxymethyl ethers, 4-pentenyl-oxymethyl (POM-OR) ethers,silyloxymethyl ethers, 2-methoxyethoxy-methyl (MEM-OR) ethers,2,2,2-trichloroethoxymethyl ethers, bis(2-chloroethoxy)-methyl ethers,2-(trimethylsilyl)ethoxymethyl (SEM-OR) ethers, methoxy-methyl (MM-OR)ethers.

[0147] Examples of protective groups (PG) of the substituted ethyl ethertype, which may be mentioned, are: 1-ethoxyethyl (EE-OR) ethers,1-(2-chloroethoxy)ethyl (Cee-OR) ethers,1-[2-(trimethylsilyl)ethoxy]ethyl (SEE-OR) ethers,1-methyl-1-methoxyethyl (MIP-OR) ethers, 1-methyl-1-benzyloxyethyl(MBE-OR) ethers, 1-methyl-benzyloxy-2-fluoro-ethyl ethers,1-methyl-1-phenoxy-ethyl ethers, 2,2,2-trichloroethyl ethers,1,1-dianisyl-2,2,2-trichloroethyl (DATE-OR) ethers,1,1,1,3,3,3-hexafluoro-2-phenyliso-propyl (HIP-OR) ethers,2-trimethylsilylethyl ethers, 2-(benzylthio)ethyl ethers,2-(phenyl-selenyl)ethyl ethers. Further examples of protective groups(PG) of the ether type, which may be mentioned, are: tetrahydropyranyl(THP-OR) ethers, 3-bromo-tetrahydropyranyl (3-BrTHP-OR) ethers,tetrahydrothiopyranyl ethers, 1-methoxy-cyclohexyl ethers, 2- and4-picolyl ethers, 3-methyl-2-picolyl-N-oxido ethers, 2-quinolinylmethyl(Qm-OR) ethers, 1-pyrenylmethyl ethers, dipenylmethyl (DPM-OR) ethers,para,para′-dinitrobenzhydryl (RO-DNB) ethers, 5-dibenzosuberyl ethers,triphenylmethyl (Tr-OR) ethers, α-naphthyldiphenyhnethyl ethers,para-methoxy-phenyldiphenylmethyl (MMTr-OR) ethers,di(para-methoxy-phenyl)phenylmethyl (DMTr-OR) ethers,tri(para-methoxy-phenyl)methyl (TMTr-OR) ethers,4-(4′-bromo-phenacyloxy) phenyldiphenylmethyl ethers,4,4′,4″-tris(4,5-dichlorophthalimido-phenyl)methyl (CPTr-OR) ethers,4,4′,4″-tris(levulinoyloxy-phenyl)methyl (TLTr-OR) ethers,4,4′,4″-tris(benzoyloxyphenyl)-methyl (TBTr-OR) ethers,4,4′-dimethoxy-3″-[N-(imidazolylmethyl)]-trityl (IDTr-OR) ethers,4,4′-dimethoxy-3″-[N-(imidazolylethyl)carbamoyl]trityl (IETr-OR) ethers,1,1-bis(4-methoxy-phenyl)-1′-pyrenylmethyl (Bmpm-OR) ethers, 9-anthrylethers, 9-(9-phenyl)xanthenyl (pixyl-OR) ethers,9-(9-phenyl-10-oxo)anthryl (tritylon ethers).4-Methoxy-tetrahydropyranyl (MTHP-OR) ethers,4-methoxy-tetrahydrothio-pyranyl ethers,4-methoxy-tetrahydrothio-pyranyl ether S,S-dioxide,1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP-OR) ethers,1-(2-fluorophenyl)-4-methoxy-piperidin-4-yl (Fpmp-OR) ethers,1,4-dioxan-2-yl ethers, tetrahydrofuranyl ethers, tetrahydrothiofuranylethers,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanebenzofuran-2-yl(RO-MBF) ethers, tert-butyl ethers, allyl ethers, propargyl ethers,para-chlorophenyl ethers, para-methoxyphenyl ethers, para-nitrophenylethers, 2,4-dinitro-phenyl (RO-DNP) ethers,2,3,5,6-tetrafluoro-4-(trifluoromethyl)phenyl ethers, benzyl (Bn-OR)ethers. Examples of protective groups of the substituted benzyl ethertype, which may be mentioned, are: para-methoxybenzyl (MPM-OR) ethers,3,4-dimethoxy-benzyl (DMPM-OR) ethers, ortho-nitrobenzyl ethers,para-nitrobenzyl ethers, para-halobenzyl ethers, 2,6-dichloro-benzylethers, para-cyanobenzyl ethers, para-phenyl-benzyl ethers,2,6-difluorobenzyl ethers, para-aminoacylbenzyl. (PAB-OR) ethers,para-azidobenzyl (Azb-OR) ethers, 4-azido-3-chlorobenzyl ethers,2-trifluoromethyl-benzyl ethers, para-(methylsulfinyl)benzyl (Msib-OR)ethers. Examples of protective groups (PG) of the silyl ether type,which may be mentioned are: trimethylsilyl (TMS-OR) ethers,triethylsilyl (TES-OR) ethers, triiso-propylsilyl (TIPS-OR) ethers,dinethylisopropyl-silyl (IPDMS-OR) ethers, diethylisopropylsilyl(DEIPS-OR) ethers, dimethylhexylsilyl (TDS-OR) ethers,tert-butyldimethylsilyl (TBDMS-OR) ethers, tert-butyldiphenylsilyl(TBDPS-OR) ethers, tribenzylsilyl ethers, tri-para-xylylsilyl ethers,triphenylsilyl (TPS-OR) ethers, diphenylmethylsilyl (DPMS-OR) ethers,di-tert-butylmethylsilyl (DTBMS-OR) ethers, tris(trimethylsilyl)silylethers (sisyl ether), (2-hydroxystyryl)-dimethylsilyl (HSDMS-OR) ethers,(2-hydroxystyryl)diisopropylsilyl (HSDIS-OR) ethers,tert-butylmethoxyphenylsilyl (TBMPS-OR) ethers, tert-butoxydiphenylsilyl(DPTBOS-OR) ethers. Examples of protective groups (PG) of the-estertype, which may be mentioned, are: formic esters, benzoylformic esters,acetic esters (RO-Ac), chloroacetic esters, dichloroacetic esters,trichloroacetic esters, trifluoroacetic esters (RO-TFA), methoxy-aceticesters, triphenylmethoxyacetic esters, phenoxyacetic esters,para-chlorophenoxy-acetic esters, phenylacetic esters, diphenylaceticesters (DPA-OR), nicotinic esters, 3-phenylpropionic esters, 4-pentenoicesters, 4-oxopentanoic esters (levulinates) (Lev-OR),4,4-(ethylenedithio)-pentanoic esters (RO-LevS),5-[3-bis(4-methoxyphenyl)hydroxy-methylphenoxy]-levulinic esters,pivalic esters (Pv-OR), 1-adamantanecarboxylic esters, crotonic esters,4-methoxy-crotonic esters, benzoic esters (Bz-OR), para-phenyl-benzoicesters, 2,4,6-trimethylbenzoic esters (mesitoic esters),4-(methylthiomethoxy)-butyric esters (MTMB-OR),2-(methylthiomethoxymethyl)benzoic esters (MTMT-OR). Examples ofprotective groups (PG) of the ester type, which may be mentioned, are:methyl carbonate, methoxymethyl carbonate, 9-fluorenylmethyl carbonate(Fmoc-OR), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc-OR),1,1-dimethyl-2,2,2-trichloro-ethyl carbonate (TCBOC-OR),2-(trimethylsilyl)ethyl carbonate (TMSEC-OR), 2-(phenylsulfonyl)-ethylcarbonates (Psec-OR), 2-(triphenylphosphonio)-ethyl carbonates(Peoc-OR), tert-butyl carbonate (Boc-OR), isobutyl carbonate, vinylcarbonate, allyl carbonate (Alloc-OR), p-nitrophenyl carbonate, benzylcarbonate (Z-OR), para-methoxybenzyl carbonate, 3,4-dimethoxybenzylcarbonate, ortho-nitrobenzyl carbonate, para-nitrobenzyl carbonate,2-dansylethyl carbonate (Dnseoc-OR), 2-(4-nitrophenyl)-ethyl carbonate(Npeoc-OR), 2-(2,4-dinitrophenyl)ethyl carbonate (Dnpeoc-OR). Examplesof protective groups (PG) of the sulfonate type, which may be mentioned,are: allylsulfonate (Als-OR), methanesulfonates (Ms-OR),benzylsulfonates, tosylates (Ts-OR), 2-[(4-nitrophenyl)ethyl]sulfonates(Npes-OR).

[0148] When preparing further spinosyn derivatives from theabovementioned compounds of the general formula (I), it may be quiteadvantageous to block initially the 1-hydroxy-ethyl radical in the C-21position and, where appropriate, the hydroxyl group in the C-9 position(where R²═H) with suitable, for example one of the abovementioned,protective groups (PG). The use of two different protective groups (PG¹and PG², respectively) is recommended here, and these protective groupsshould be appropriately compatible, i.e. removable from one anotherselectively and independently. Subsequently, a derivatization, forexample glycosidation, by means of chemical synthesis or by microbialbioconversion (cf. also U.S. Pat. No. 5,539,089; introduction of theradicals R or R′) may take place on the hydroxyl group in the C-17position to give the spinosyn derivatives (Ia-2 or Ib-2) (cf. schemes 2and 3).

[0149] After successful derivatization in positions. C-9 and,respectively, C-17 (cf Ia-4) or C-17 (cf. Ib-2), the remainingprotective group (PG) can be removed and the spinosyn derivative of thegeneral formula (D) be obtained.

EXAMPLES Example 1

[0150] Strains Used

[0151] Table: Strains capable of biotransformation of the compound (IIa)to give the corresponding derivatives having a 1-hydroxy-ethyl radicalin the C-21 position (compound (Ia)): Name Internal reference DepositionNo. Streptomyces djakartensis NRRL B-12103 DSM 14327 Streptomycesgriseofuscus DSM 40191 DSM 14330 Streptomyces caelestis DSM40084 DSM14328 Streptomyces antibioticus ATCC 11891 DSM 14329 Streptomycesgriseus DSM 40937 DSM 14331 Streptomyces aureofaciens DSM 46447 DSM14332

Example 2

[0152] Biotransformation Using S. djakartensis NRRL B-12103

[0153] Exemplary biotransformation protocol for producing the compound(Ia) from the compound (IIa).

[0154] The producer cultures were prepared by inoculating 20 ml of R5Amedium (per liter of R5A medium: 103 g of sucrose; 0.25 g of K₂SO₄;10.12 g of MgCl₂; 10 g of glucose; 5 g of yeast extract; 0.1 g ofcasamino acids, 21 g of MOPS buffer pH 6:8 (KOH), 2 ml of trace elementsolution; trace element solution: (per liter) 40 mg of ZnCl₂, 200 mg ofFeCl₃×6H₂O, 10 mg of CuCl₂×2H₂O, 10 mg of MnCl₂×4H₂O, 10 mg ofNa₂B₄O₇×10H₂O, 10 mg of (NH₄)₆Mo₇O₂₄×4H₂O; (Fernandez et al., 1998, J.Bacteriol. 180: 4929; Hopwood et al., 1985, Genetic manipulation ofStreptomyces. A laboratory manual. The John Innes Foundation, Norwich,England) in 100 ml Erlenmeyer flasks with in each case 50 μl of S.djakartensis NRRL B-12103 spore suspension. Prior to inoculation, themedium was sterilized at 121° C. and an overpressure of 1.1 for 20minutes. The cultures were incubated at 28° C. and 200 rpm. After 24hours and after 72 hours of incubation, in each case 1 mg of thecompound (IIa) (100 μl of a stock solution of 10 mg/ml in methanol) wasadded. The biotransformation was stopped after 120 hours. The cultureswere removed by centrifugation (4000 rpm, 10 minutes) and thesupernatant was admixed with the same volume of methanol.

Example 3

[0155] Biotransformations Using Streptomyces griseofuscus

[0156] Exemplary biotransformation protocol for producing the compound(Ia) from the compound (IIa).

[0157] The producer cultures were prepared by applying the method ofExample 2, using 50 μl of spore suspension of the strain Streptomycesgriseofuscus instead of the strain S. djakartensis NRRL B-12103.

Example 4

[0158] Biotransformations Using Streptomyces caelestis

[0159] Exemplary biotransformation protocol for producing the compound(Ia) from the compound (IIa).

[0160] The producer cultures were prepared by applying the method ofExample 2, using 50 μl of spore suspension of the strain Streptomycescaelestis instead of the strain S. djakartensis NRRL B-12103.

Example 5

[0161] Biotransformations Using Streptomyces antibioticus

[0162] Exemplary biotransformation protocol for producing the compound(Ia) from the compound (IIa).

[0163] The producer cultures were prepared by applying the method ofExample 2, using 50 μl of spore suspension of the strain Streptomycesantibioticus instead of the strain S. djakartensis NRRLB-12103.

Example 6

[0164] Biotransformations Using Streptomyces griseus

[0165] Exemplary biotransformation protocol for producing the compound(Ia) from the compound (IIa).

[0166] The producer cultures were prepared by applying the method ofExample 2, using 50 μl of spore suspension of the strain Streptomycesgriseus instead of the strain S. djakartensis NRRL B-12103.

Example 7

[0167] Biotransformations Using Streptomyces aureofaciens

[0168] Exemplary biotransformation protocol for producing the compound(Ia) from the compound (IIa).

[0169] The producer cultures were prepared by applying the method ofExample 2, using 50 μl of spore suspension of the strain Streptomycesaureofaciens instead of the strain S. djakartensis NRRL B-12103.

Example 8

[0170] Biotransformations Using Streptomyces djakartensis

[0171] Exemplary biotransformation protocol for producing spinosyn Awith a 1-hydroxy-ethyl radical in the C-21 position [compound of thegeneral formula (I) in which R¹ is an amino sugar of the formula Ia, A-Bis the group —HC═CH— and D is the group —CO—R² where R² is a sugar ofthe formula 2a].

[0172] The producer cultures were prepared by inoculating 20 ml of R5Amedium (R5A medium: see Example 2) in 100 ml Erlenmeyer flasks with ineach case 50 μl of S. djakartensis NRRL B-12103 spore suspension. Priorto inoculation, the medium was sterilized at 121° C. and an overpressureof 1.1 bar for 20 minutes. The cultures were incubated at 28° C. and 200rpm. After 48 hours of incubation, 2 mg of spinosyn A (100 μl of a stocksolution, of 10 mg/ml in methanol) were added. The biotransformation wasstopped after 96 hours. The cultures were removed by centrifugation(4000 rpm, 10 minutes)-and the supernatant was admixed with the samevolume of methanol.

Example 9

[0173] Biotransformations Using Streptomyces djakartensis

[0174] Exemplary biotransformation protocol for producing17-pseudo-spinosyn aglycone A with a 1-hydroxy-ethyl radical in the C-21position [compound of the general formula (I) in which R¹ is hydrogen,A-B is the group —HC═CH— or —HC═C(CH₃)— and D is the group —CO—R² whereR² is a sugar of the formula 2a].

[0175] The producer cultures were prepared by inoculating 20 ml of R5Amedium (R5A medium: see Example 2) in 100 ml Erlenmeyer flasks with ineach case 50 A1 of S. djakartensis NRRL B-12103 spore suspension. Priorto inoculation, the medium was, sterilized at 121° C. and anoverpressure of 1.1 bar for 20 minutes. The cultures were incubated at28° C. and 200 rpm. After 48 hours of incubation, 2 mg of17-pseudospinosyn aglycone A or D (100 μl of a stock solution of 10mg/ml in methanol) were added. The biotransformation was stopped after96 hours. The cultures were removed by centrifugation (4000 rpm, 10minutes) and the supernatant was admixed with the same volume ofmethanol.

Example 10

[0176] Isolation of the Compound (Ia) from the Biotransformation with S.djakartensis NRRL B-12103

[0177] Exemplary protocol for working up the culture supernatants andconcentrating the compound (Ia).

[0178] 35 ml of the culture supernatant of Example 2, to-which methanolhad been added, were reduced to about 20 ml and admixed with 10 ml ofwater. This was followed by extracting twice with in each case 10 ml ofethyl acetate, concentrating the combined organic phases to dryness andresuspending the residue in 400 μl of methanol. An aliquot of thissolution was analyzed via HPLC/MS (Example 11).

Example 11

[0179] Analytical HPLC/UV/MS

[0180] Exemplary protocol for analyzing the worked-up culturesupernatants by means of HPLC/UV/MS.

[0181] An aliquot of the worked-up culture, supernatant of thebiotransformation with S. djakartensis (Example 2) were subjected tochromatography on a reversed-phase HPLC column (2.1×250 mm) with agradient of water to which ammonium acetate (25 mmol/1) had been addedand methanol to which ammonium acetate (25 mmol/l) had been added and aflow rate of 250 p/minute. The detection is carried out using UV (245nm) and electrospray (positive) mass spectrometry on a quadrupole massspectrometer.

[0182] Compound (Ia) has a molecular weight of 418 Dalton and isdetected as [M+NH₄]⁺ ion at m/z=436 under these conditions. Theretention time of approx. 32.5 minutes is shorter than that of thecompound (IIa), which is approx. 37.5 minutes.

Example 12

[0183] Extraction and Preparative Preparation in Pure Form of theCompound (Ia) from Shaker Cultures of the Biotransformation with S.djakartensis NRRL B-12103

[0184] Fifteen 20 ml cultures of the strain (S. djakartensis) were grownin 100 ml Erlenmeyer flasks according to the method described in Example2 and the culture supernatants were combined to work up compound (Ia).The combined culture supernatants were worked up as described in Example11. The residue was resuspended in about 3 ml of methanol. The compound(Ia) was isolated via chromatography on an analytical reversed-phaseHPLC column (4.6×250 mm) with a gradient of 25 mmol/l ammonium acetatein water and 25 mmol/l ammonium acetate in methanol. An aliquot of 100μl was injected for each run. The UV-detection was carried out at 245nm. The fractions were collected manually, combined and evaporated todryness. The yield was approximately 1 mg.

Example 13

[0185] Elucidation of the Structure of the Compound (Ia)

[0186] The preparatively isolated compound (Ia) was resuspended in CD₃ODand studied by nuclear magnetic resonance (NMR). ¹H-NMR, COSY, TOCSY,HSQC and HMBC spectra were recorded. The table below summarizes theresults.

[0187] NMR data of compound (Ia) in CD₃OD (500 MHz) δ_(C) δ_(H) Position[ppm] [ppm] Mult. J [Hz] Int. 1 174 — — — 2 35 2.48 dd 14/3 1H 3.12 dd14/5 1H 3 49 2.93 ddd 10/5/2 1H 4 43 3.49 m 1H 5 129 5.84 ddd 10/3/3 1H6 131 5.91 d br. 10 1H 7 42 2.23 m 1H 8 41 1.46 m 1H 1.83 dd 13/7 1H 973 4.34 m 1H 10 40 1.24 m 1H 2.34 m 1H 11 47 0.94 m 1H 12 51 2.85 m 1H13 150 7.00 s br. 1H 14 146 — — 15 206 — — 16 50 3.22 m 1H 17 73 3.49 m1H 18 36 1.43 m 1H 1.48 m 1H 19 23 1.28 m 1H 1.71 m 1H 20 27 1.48 m 1H1.63 m 1H 21 79 4.70 ddd 10/5/2 1H 22 70 3.65 m 1H 23 18 1.04 d 7 3H 2416 1.15 d 7 3H (Ia)

Examples 14

[0188] Analytical Detection of the Hydroxylated Spinosyn A Produced

[0189] 20 ml of the culture supernatant of the biotransformation ofExample 8 to which methanol had been added, were adjusted to pH 5 with a0.01 N NaOH solution and concentrated to approx. 5 ml of aqueous residuewhich was then extracted twice with in each case 5 ml of ethyl acetate.The combined organic phases were concentrated to dryness in an N₂ streamand resuspended in 200 μl of methanol. An aliquot of this extract wasstudied by means of LC/MS and LC/MS/MS on a tandem mass spectrometerwith electrospray positive ionization.

[0190] In the LC/MS chromatogram of the extract a peak appears at 40.0minutes with [M+H]⁺ m/z=748.5 at low concentration. The spectrum of thedaughter ions of this ion has a fragment with m/z=142 which ischaracteristic for the removal of a forosamine unit.

Examples 15

[0191] Analytical Detection of the Hydroxylated 17-pseudo-spinosynAglycone Produced

[0192] 20 ml of the culture supernatant of the biotransformation ofExample 9 to which methanol had been added, were adjusted to pH 5 with a0.01 N NaOH solution and concentrated to approx. 5 ml of aqueous residuewhich was then extracted twice with in each case 5 ml of ethyl acetate.The combined organic phases were concentrated to dryness in an N₂ streamand resuspended in 200 μl of methanol. An aliquot of this extract wasstudied by means of LC/MS and LC/MS/MS on a tandem mass spectrometerwith electrospray positive ionization.

[0193] In the LC/MS chromatogram of the extract a peak appears at 38.8minutes with [M+NH₄]⁺ m/z=624.4 at low concentration. This correspondsto a hydroxylated product of 17-pseudo-spinosyn A aglycone. The spectrumof the daughter ions of this ion has a fragment with m/z=189 which ischaracteristic for the removal of a trimethylrhamnose unit.

Example 16

[0194] Characterization of the Strains Used

[0195] a) Streptomyces djakartensis NR B-12103:

[0196] This strain was obtained as a niddamycin producer from theAgricultural Research Service Culture Collection (1815 N. UniversityStreet, Illinois 61604, U.S.A.) with accession number NRRL B-12103. Thestrain is described in US 3646194. The culture was deposited again withthe Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ),Mascheroder Weg 1b, D-38124 Brunswick, Germany with deposition numberDSM 14327 in accordance with the requirements of the Budapest treaty on06.06.2001.

[0197] b) Streptomyces griseofuscus DSM 40191:

[0198] This strain was obtained as bundlin A, B, moldicidin A andpentarnycin producer from the Deutsche Sammlung von Mikroorganismen undZellkulturen (Mascheroder Weg 1b, D-38124 Brunswick, Germany) withaccession number 40191. The culture was deposited again with theDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ),Mascheroder Weg 1b, D-38124 Brunswick, Germany, with deposition number,DSM 14330 in accordance with the requirements of the Budapest treaty on06.06.2001.

[0199] c) Streptomyces caelestis DSM 40084:

[0200] This strain was obtained as caelesticetin producer from theDeutsche Sammlung von Mikroorganismen und Zellkulturen (Mascheroder Weg1b, D-38124 Brunswick, Germany) with accession number 40084. The culturewas deposited again with the Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Brunswick,Germany, with deposition number DSM 14328 in accordance with therequirements of the Budapest treaty on 06.06.2001.

[0201] d) Streptomyces antibioticus ATCC 11891:

[0202] This strain was obtained as a caelesticetin producer from theAmerican Type Culture Collection (10801 University Boulevard, Manassas,Va. 20110-2209, USA) with accession number ATCC 11891. The culture wasdeposited again with the Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Brunswick,Germany, with deposition number DSM 14329 in accordance with therequirements of the Budapest treaty on 06.06.2001.

[0203] e) Streptomyces griseus DSM 40937:

[0204] This strain was obtained from the Deutsche Sammlung vonMikroorganismen und Zellkulturen (Mascheroder Weg 1b, D-38124 Brunswick,Germany) with accession number 40937. The culture was deposited againwith the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Mascheroder Weg 1b, D-38124 Brunswick, Germany, with depositionnumber DSM 14331 in accordance with the requirements of the Budapesttreaty on 06.06.2001.

[0205] f) Streptomyces aureofaciens DSM 46447:

[0206] This strain was obtained as tetracycline producer from theDeutsche Sammlung von Mikroorganismen und Zellkulturen (Mascheroder Weg1b, D-38124 Brunswick, Germany) with accession number 40084. The culturewas deposited again with the Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Brunswick,Germany, with deposition number DSM 14332 in accordance with therequirements of the Budapest treaty on 06.06.2001.

Example 17

[0207] Preparation of the Starting Compounds

[0208] Spinosyn A Aglycone (IIa)

[0209] The spinosyn A aglycone (IIa) [compound of the general formula(II) in which R¹ is hydrogen, A-B is the group —HC═CH— and D is thegroup C—OH] was prepared from Tracer® as described in WO 01/16303.

[0210] 9-Keto-Spinosyn A Aglycone (IIb)

[0211] The 9-keto-spinosyn A aglycone (IIb) [compound of the generalformula (II) in which R¹ is hydrogen, A-B is the group —HC═CH— and D isthe group C═O] was prepared from the compound (IIa) by means ofpyridinium dichromate oxidation:

[0212] 46.55 g (115.6 mmol) of the spinosyn A aglycone (1Ha) weredissolved in 1100 ml of abs. dichloromethane under inert gas and admixedwith 43.51 g (115.6 mmol) of pyridinium dichromate. After stirring at25° C. for 4 hours and addition of 900 ml of diethyl ether, theprecipitated chromium salts were filtered off and the filtrate wasconcentrated under reduced pressure. Column chromatography on silica gel(eluent: cyclohexane/ethyl acetate 1:1, followed by 100% ethyl acetate)delivered 3.68 g of 9,17-diketospinosyn aglycone and 23.40 g of anapprox. 9:1 mixture of the spinosyn A aglycone (IIa) and the17-keto-spinosyn aglycone (IIb) in addition to 11.74 g of recoveredspinosyn A aglycone (IIa). Recrystallization of the mixture incyclohexane/ethyl acetate concentrates the 9-keto-spinosyn A aglycone(11b) to >98%. 20.78 g of 9-keto-spinosyn A aglycone (IIb) are obtainedin the form of colorless crystals.

[0213] TLC: R_(f) (SiO₂, ethyl acetate=0.44—¹H-NMR: CDCl₃, δ =6.77 (s,13-H); 5.97 (d, 6-H); 5.88 (m, 5-H); 4.72 (m, 21-H); 3.69 (m, 17-H)inter alia—LC/ESI-MS: m/z=401 (25%) [M]⁺, 289 (100%).

[0214] Diketospinosyn aglycone: TLC: R_(f) (SiO₂, ethylacetate)=0.64—¹H-NMR: CDCl₃, δ =6.92 (s, 13-H); 5.97 (d, 6-H); 5.87 (m,5-H); 4.85 (m, 21-H); 4.25 (q, 16-H) inter alia—LC/ESI-MS: m/z=399(100%) [M+H]⁺.

[0215] Spinosyn A

[0216] The preparation of spinosyn A was initially carried out asdescribed in WO 01/16303. The spinosyn A/D mixture obtained wasfractionated by chromatography on a preparative reversed-phase column(250×8 mm). The eluents used were water containing 25 mmol/l ammoniumacetate (A) and methanol containing 25 mmol/l ammonium acetate (B).Elution was carried out using a gradient of from 60% B to 100% B in 35minutes. The flow rate was 3 ml/minute. The separated substances weredetected by a TV detector at 242 nm and automatically fractionated.Spinosyn A eluted at approx. 31 minutes and spinosyn D at approx. 33minutes. The combined fractions of spinosyn A from several injectionswere evaporated down to the aqueous residue under reduced pressure inthe rotary evaporator. Said aqueous solution was freeze-dried andspinosyn A was obtained as a white solid.

[0217] 17-Pseudospinosyn A/D Aglycone

[0218] The 17-pseudospinosyn A/D aglycone was prepared as described inWO 01/16303.

1. A method for producing a compound of the formula (I),

in which A-B is any of the following groups: —HC═CH—, —HC═C(CH₃)—,—H₂C—CH₂— or —H₂C—CH(CH₃)—, D is the group

R¹ is hydrogen or an amino sugar and R² is hydrogen or a sugar,comprising contacting a compound of the formula (II),

in which A-B, D and R¹ are as defined above, with a microorganism in anaqueous nutrient medium under aerobic conditions or with an enzymeextract prepared therefrom or with one or more enzymes isolatedtherefrom.
 2. The method as claimed in claim 1, wherein in the case (1)that in the compound of the formula (II) A-B is any of the followinggroups: —HC═CH—, —HC═C(CH₃)—, —H₂C—CH₂— or —H₂C—CH(CH₃)— and D is thegroup

R¹ is an amino sugar of the formula 1a

and R² is a sugar of the formula 2a

or in the case (2) that in the compound of the formula (II) A-B is thegroup —HC═CH— or —H₂C—CH₂— and D is as defined above, R¹ is an aminosugar of the abovementioned formula 1a and R² is hydrogen or a sugar ofthe formula 2b, 2c, 2d, 2e or 2f

or in the case (3) that in the compound of the formula (II) A-B is anyof the following groups: —HC═CH—, —HC═C(CH₃)— or —H₂C—CH₂— and D is asdefined above, R¹ is hydrogen or an amino sugar of the formula 1b

and R² is hydrogen or a sugar of the abovementioned formula 2a or in thecase (4) that in the compound of the formula (II) A-B is the group—HC═CH— or —HC═C(CH₃)— and D is as defined above, R¹ is an amino sugarof the abovementioned formula 1a and R² is a sugar of the formula 2g,2h, 2i, 2j or 2k

or in the case (5) that in the compound of the formula (II) A-B is thegroup —HC═CH— or —H₂C—CH₂— and D is as defined above, R¹ is an aminosugar of the abovementioned formula 1a and R² is a sugar of the formula2l or 2m

or in the case (6) that in the compound of the formula (II) A-B is thegroup —HC═CH— or —HC═C(CH₃)— and D is as defined above, R¹ is hydrogenor an amino sugar of the abovementioned formula 1b or an amino sugar ofthe formula 1c

and R² is a sugar of the abovementioned formula 2b, 2c, 2g or 2h or asugar of the formula 2n

or in the case (7) that in the compound of the formula (II) A-B is thegroup —HC═CH— and D is as defined above, R¹ is an amino sugar of theabovementioned formula 1a and R² is a sugar of the formula 2o

or in the case (8) that in the compound of the formula (II) A-B is thegroup —HC═CH— and D is as defined above, R¹ is an amino sugar of theabovementioned formula 1b and R² is a sugar of the abovementionedformula 2d, 2i or 2j or a sugar of the formula 2p

or in the case (9) that in the compound of the formula (II) A-B is thegroup —HC═CH— and D is as defined above, R¹ is hydrogen or an aminosugar of the abovementioned formula 1c and R² is a sugar of theabovementioned formula 2i or 2p or in the case (10) that in the compoundof the formula (II) A-B is the group —HC═CH— and D is as defined above,R¹ is an amino sugar of the formula 1d, 1e or 1f

and R² is a sugar of the abovementioned formula 2a or in the case (11)that in the compound of the formula (II) A-B is the group —HC═CH— and Dis the group

R¹ is hydrogen or an amino sugar of the abovementioned formula 1a. 3.The method as claimed in claim 1 wherein in the case (12) that in thecompound of the formula (II) A-B is the group —HC═CH— or —HC═C(CH₃)— andD is the group

R¹ is an amino sugar of the formula 1a and R² is a sugar of the formula2a, 2g or 2h or in the case (13) that in the compound of the formula(II) A-B is the group —HC═CH— and D is as defined above, R¹ is an aminosugar of the formula 1a and R² is hydrogen or a sugar of the formula 2d,2e, 2l, 2m or 2o or in the case (14) that in the compound of the formula(II) A-B is the group —HC═CH— or —HC═C(CH₃)— and D is as defined above,R¹ is hydrogen or an amino sugar of the formula 1b and R² is hydrogen ora sugar of the formula 2a or in which A-B, D and R¹ are as defined as inthe case (11).
 4. The method as claimed in claim 1, wherein in the case(15) that in the compound of the formula (II) A-B is the group —HC═CH—or —HC═C(CH₃)— and D is the group

R¹ is an amino sugar of the formula 1a and R² is a sugar of the formula2a or in the case (16) that in the compound of the formula (II) A-B isthe group —HC═CH— and D is as defined above, R¹ is an amino sugar of theformula 1a and R² is hydrogen or a sugar of the formula 2d, 2l or 2m orin which A-B, D and R¹ are as defined in the case (11).
 5. The method asclaimed in claim 1, wherein in the case (17) that in the compound of theformula (II) A-B is the group —HC═CH— or —HC═C(CH₃)— and D is the group

R¹ is an amino sugar of the formula 1a and R² is a sugar of the formula2a or in the case (18) that in the compound of the formula (II) A-B isthe group —HC═CH— and D is as defined above, R¹ is hydrogen and R² ishydrogen.
 6. The method as claimed in claim 1 wherein the microorganismis a microorganism selected from the group of actinomycetes.
 7. Themethod as claimed in claim 6, wherein the microorganism selected fromthe group of actinomycetes is a microorganism of the genus Streptomyces.8. The method as claimed in claim 7, wherein the microorganism of thegenus Streptomyces is a strain of the species selected from the groupconsisting of Streptomyces djakartensis, Streptomyces griseofuscus,Streptomyces caelestis, Streptomyces antibioticus, Streptomyces griseusand Streptomyces aureofaciens.
 9. The method as claimed in claim 8,wherein the strain has the characteristic features of the strainsStreptomyces djakartensis DSM 14327, Streptomyces griseofuscus DSM14330, Streptomyces caelestis DSM 14328, Streptomyces antibioticus DSM14329, Streptomyces griseus DSM 14331 or Streptomyces aureofaciens DSM14332.
 10. The method as claimed in claim 1 wherein the compound of theformula (I) is isolated.
 11. A compound of the formula (I),

in which A-B is any of the following groups: —HC═CH—, —HC═C(CH₃)—,—H₂C—CH₂— or —H₂C—CH(CH₃)—, D is the group

R¹ is an amino sugar and R² is a sugar.
 12. The compound as claimed inclaim 11, wherein in the case (1) that in the compound of the formula(I) A-B is any of the following groups: —HC═CH—, —HC═C(CH₃)—, —H₂C—CH₂—or —H₂C—CH(CH₃)— and D is the group

R¹ is an amino sugar of the formula 1a

and R² is a sugar of the formula 2a

or, in the case (2) that in the compound of the formula (I) A-B is thegroup —HC═CH— or —H₂C—CH₂— and D is as defined above, R¹ is an aminosugar of the abovementioned formula 1a and R² is hydrogen or a sugar ofthe formula 2b, 2c, 2d, 2e or 2f

or, in the case (3) that in the compound of the formula (I) A-B is oneof the following groups: —HC═CH—, —HC═C(CH₃)— or —H₂C—CH₂— and D is asdefined above, R¹ is hydrogen or an amino sugar of the formula 1b

and R² is hydrogen or a sugar of the abovementioned formula 2a or, inthe case (4) that in the compound of the formula (I) A-B is the group—HC═CH— or —HC═C(CH₃)— and D is as defined above, R¹ is an amino sugarof the abovementioned formula 1a and R² is a sugar of the formula 2g,2h, 2i, 2j or 2k

or, in the case (5) that in the compound of the formula (I) A-B is thegroup —HC═CH— or —H₂C—CH₂— and D is as defined above, R¹ is an aminosugar of the abovementioned formula 1a and R² is a sugar of the formula2l or 2m

or, in the case (6) that in the compound of the formula (1) A-B is thegroup —HC═CH— or —HC═C(CH₃)— and D is as defined above, R¹ is hydrogenor an amino sugar of the abovementioned formula 1b or an amino sugar ofthe formula 1c

and R² is a sugar of the abovementioned formula 2b, 2c, 2g or 2h or asugar of the formula 2n

or, in the case (7) that in the compound of the formula (I) A-B is thegroup —HC═CH— and D is as defined above, R¹ is an amino sugar of theabovementioned formula 1a and R² is a sugar of the formula 2o

or, in the case (8) that in the compound of the formula (I) A-B is thegroup —HC═CH— and D is as defined above, R¹ is an amino sugar of theabovementioned formula 1b and R² is a sugar of the abovementionedformula 2d, 2i or 2j or a sugar of the formula 2p

or, in the case (9) that in the compound of the formula (I) A-B is thegroup —HC═CH— and D is as defined above, R¹ is hydrogen or an aminosugar of the abovementioned formula 1c and R² is a sugar of theabovementioned formula 2i or 2p or, in the case (10) that in thecompound of the formula (I) A-B is the group —HC═CH— and D is as definedabove, R¹ is amino sugar of the abovementioned formula 1d, 1e or anamino sugar of the formula 1f

and R² is a sugar of the abovementioned formula 2a or, in the case (11)that in the compound of the formula (I) A-B is the group —HC═CH— and Dis the group

—R¹ is hydrogen or an amino sugar of the abovementioned formula 1a. 13.The compound as claimed in claim 12, wherein in the case (12) that inthe compound of the formula (I) A-B is the group —HC═CH— or —HC═C(CH₃)—and D is the group

R¹ is an amino sugar of the formula 1a and R² is a sugar of the formula2a, 2g or 2h or, in the case (13) that in the compound of the formula(I) A-B is the group —HC═CH— and D is as defined above, R¹ is an aminosugar of the formula 1a and R² is hydrogen or a sugar of the formula 2d,2e, 2l, 2m or 2o or, in the case (14) that in the compound of theformula (I) A-B is the group —HC═CH— or —HC═C(CH₃)— and D is as definedabove, R¹ is hydrogen or an amino sugar of the formula 1b and R² ishydrogen or a sugar of the formula 2a or A-B, D and R¹ are as defined asin the case (11).
 14. The compound as claimed in claim 12, wherein inthe case (15) that in the compound of the formula (I) A-B is the group—HC═CH— or —HC═C(CH₃)— and D is the group

R¹ is an amino sugar of the formula 1a and R² is a sugar of the formula2a or, in the case (16) that in the compound of the formula (I) A-B isthe group —HC═CH— and D is as defined above, R¹ is an amino sugar of theformula 1a and R² is hydrogen or a sugar of the formula 2d, 2l or 2m orA-B, D and R¹ are as defined as in the case (11).
 15. The compound asclaimed in claim 12, wherein in the case (17) that in the compound ofthe formula (I) A-B is the group —HC═CH— or —HC═C(CH₃)— and D is thegroup

R¹ is an amino sugar of the formula 1a and R² is a sugar of the formula2a or, in the case (18) that in the compound of the formula (I) A-B isthe group —HC═CH— and D is as defined above, R¹ is hydrogen and R² ishydrogen.
 16. The compound as claimed in claim 12, wherein in thecompound of the formula (I) A-B is the group —HC═CH—, D is the group

R¹ is an amino sugar of the formula 1a and R² is a sugar of the formula2a, or in which in the compound of the formula (I) A-B is the group—HC═CH— or —HC═C(CH₃)—, D is as defined above, R¹ is hydrogen and R² isa sugar of the formula 2a, or in which in the compound of the formula(I) A-B is a group —HC═CH—, D is as defined above, R¹ is hydrogen and R²is hydrogen.
 17. (Cancelled)